The term refraction refers to the change in direction of a wave due to a change in the speed of the wave. We encounter the effects of refraction in our day to day life. An object in a pool is not where it appears to be when one attempts to grasp it, or a straw in a glass of water, when observed from outside the glass, appears disjoint. The effects of refraction in these contexts may have little or no practical consequence in one's daily life. However, within the context of a system that analyzes and/or measures radiated waves (e.g., light, sound, etc.), the effects of refraction are particularly important.
A cytometer is one such instrument that analyzes and/or measures radiated waves. Cytometers analyze and/or measure various parameters of the waves to count and/or classify particles or cells. For simplicity, this specification will hereafter use the term “cell,” though the principles taught and claimed herein may apply with equal force to other types of particulate matter or discrete bodies. Additionally, the term “objective lens” is used throughout the specification, in accordance with its ordinary meaning, to indicate a lens or combination of lenses that first receives the rays from an object under observation. Further, while the principles taught and claimed herein are described with respect to cytometers and, in particular, with respect to cytometers that measure and/or analyze light waves (i.e., electromagnetic waves with a wavelength between approximately 10 nm and 100 μm), these principles are applicable in any system measuring or analyzing energy exhibiting wave transmission. Still further, the following detailed description describes embodiments utilizing one or more electronic detectors, the output of which a computer or other electronic means analyzes or measures. However, the detector may be other than an electronic detector (e.g., a human eye may be a detector), and the analysis and/or measurement means need not be electronic (e.g., where a brain analyzes light detected by a human eye).
Cytometers analyze and/or measure light by collecting the light through a system of optical elements. The collected light may be light reflected, transmitted, and/or emitted by the object being observed. As just one example, an illumination source (e.g., an ultraviolet illumination source) may illuminate a cell, causing the cell, or a chemical or dye within the cell, to emit light of a different wavelength (e.g., fluorescent light). The optical elements may include lenses, mirrors, filters, and the like, that cooperate to form an optical path. The collected light follows the optical path to a detector (e.g., a photodiode, a human eye, etc.) where the light is analyzed and/or measured. In the example above, the detector may detect a peak in the received light for each cell in or passing through a detection/interrogation area, and a computer may count the peaks to determine the number of cells. Alternatively, or in addition, the detector may detect different amounts or types of light corresponding to different cells, and a computer may interpret or analyze signals received from one or more detectors.
The various optical elements through which a cytometer collects light typically include a variety of materials (e.g., glass lenses, plastic filters, crystalline materials, metallic surfaces, etc.). Moreover, in traversing the entirety of the optical path, from the origin of the light to a detector, the light may pass through any number of materials and/or environments. For example, fluorescent light emitted from a stain attached to deoxyribonucleic acid (DNA) of a cell may pass through: various materials within the cell; a cell membrane; a buffer solution and/or cell medium in which the cell is suspended and/or bathed; a cover slip or other container material; a fixing agent; water; oil; air; a glass lens, etc. Each of these materials may have different properties with regard to the light waves incident upon the material, which properties may affect the speed of the light waves through the material and, ultimately, the path of the light. In short, refraction occurring at the interfaces of the various materials in the optical path of a cytometer can alter the path of light collected to image the analyte. (Of course, refraction may also affect illumination light directed toward the analyte.) The effect of such alterations in the path of the collected (or transmitted) light may include a reduction in the peak power or intensity of incident or imaging light delivered to or emitted from the analyte in a focus series across the analyte. Similarly, power or intensity profile in a focus series may broaden, reflecting an increase in the effective focal volume for the system.
One of the properties of a material is the refractive index. The refractive index is a number that indicates the speed of light in a given medium as either the ratio of the speed of light in a vacuum to that in the given material (i.e., an absolute refractive index) or the ratio of the speed of light in a specified medium to that in the given medium (i.e., a relative refractive index). Unless otherwise specified, refractive indices within this specification are absolute refractive indices.
Solids and liquids generally have particularly large differences in their refractive indices. For example, the refractive index of water (which varies by temperature and wavelength) is in the range 1.331-1.345 at 20° C. Buffers for use in cytometry typically contain dissolved salts and other chemicals and have a refractive index similar to or higher than water alone. Such buffers are typically used to contain and/or transport cells that are the subject of the analysis (the ‘analyte’). Materials used to construct elements of the optical path, such as an optical cell, include glasses, plastics, and crystalline materials, of which some examples may include acrylic, polycarbonate, quartz, sapphire glass, polystyrene, polypropylene, and/or other materials. Each of these solid materials typically has a refractive index significantly different from (and usually greater than) that of water.
Well known to those skilled in the art of optical system design and construction are various approaches to ameliorating aberrations arising from the shape, position, and optical properties of the various elements of optical systems. Such systems include, by way of example but not exclusion, telescopic systems, microscope systems, and imaging systems. Optical system design frequently involves the selection of materials, numbers and shapes of optical elements (where the figuring of optical elements of differing complexity is associated with different costs), and configurations, where the requirements of the system are assessed against the cost of achieving optical performance that suffices to carry out the desired function. For example, a telescope that produces images for visual observation may perform satisfactorily despite the presence of chromatic aberrations induced by the different refraction angle of light of different wavelengths as it passes through the lenses of the system. However, additional optical elements may be required to reduce or eliminate chromatic aberration in a similar telescope intended for precise astronomical photography. Furthermore, as another example, in telescopes and other optical systems, specially shaped lenses may be introduced to compensate for systematic aberrations introduced in the imaging of the object of study by the use of other elements that are ground to spherical curves, an aberration known as spherical aberration. Furthermore, in yet another example, optical systems may be designed that correct for specific and well-understood aberrations that occur outside of the lenses and other conventional components of the constructed optical system. For instance, water immersion type microscope compound objective lenses are now produced that correct for aberrations in the optical path in imaging an object lying beneath a cover slip and a layer of water, where the optical system is designed to correct for the refraction of light at both sides of the cover slip. Such corrective design may offer improved focus and resolution relative to optical systems that do not correct for systematic aberrations introduced by the properties of the materials through which the imaging light passes before entering the objective lens.
The possibility of designing an optical system to compensate for aberrations that are internal to the optical system, or for aberrations that occur as a result of materials that are part of or near to the object being imaged, in no way reduces the fact, well understood to those skilled in the art, that it is desirable to reduce or eliminate such aberrations where possible. By way of example, oil immersion, where the space between the objective lens of the microscope and the cover slip of the sample is filled with an oil having a refractive index matching that of the coverslip glass, is commonly used in microscopy to reduce or eliminate the refraction that would occur at the air-coverglass interface in the absence of the oil. In practice, some aberrations that are introduced by materials and apparatus through which imaging light must be collected are not readily or affordably corrected in the design of an optical system. By way of example only, liquid jet-in-air cytometers feature a roughly cylindrical jet of aqueous fluid containing cells that are the object of study. Lenses to correct for aberrations caused by the interface of the aqueous cylinder with the surrounding air have not been developed, since the expense and technical difficulty of designing such lenses is high. Nevertheless, those skilled in the art of cytometry will appreciate that cytometers with enclosed liquid streams featuring flat transparent walls through which imaging light is collected, may feature improved imaging, signal strength, focus, and/or resolution by virtue of reduced optical aberration.
FIGS. 1 and 2 illustrate the problem that results from the boundaries between materials having different refractive indices. FIG. 1 depicts a typical microscope objective 10. The microscope objective 10 acts to focus light waves 12 passing through the microscope objective 10 to a nominal focal point (NFP) 14. FIG. 2 depicts the same microscope objective 10. A cover slip 16, such as a cover slip 16 that may be used with a microscope slide (not shown), is disposed in between the objective lens 10 and the NFP 14. The cover slip 16 is a solid material (e.g., glass or plastic) having a refractive index higher than that of a medium 18 (e.g. air) on either side of the cover slip 16. The refractive index change occurring at an interface 20 of the cover slip 16 and the medium 18, and the refractive index change occurring at an interface 22 of the cover slip 16 and the medium 18, result in a shift in the position of the NFP 14 away from the lens 10. The modified focal point is referred to as an Actual Focal Position (AFP) 24. The AFP 24 is not a point but a region or volume in space, due to aberration induced by the refractive index changes in the media. The aberration may be characterized as a point spread function for the system and may be calculated numerically. As a consequence of the aberration of the AFP 24, the peak intensity of light measured in a focus series (focusing through a sample located at a defined position) is reduced, and the full-width half maximum of the distribution is broadened.
In confocal microscopy there exists an alternative to using a specially designed optical system to mitigate the effects of refraction. U.S. Pat. No. 5,406,421 describes a coverslip for use in a confocal microscope. The coverslip is made of a transparent material having a refractive index which is lower or higher than that of water by 0.02 or less. In particular, the coverslip is made of a transparent fluorocarbon resin having a refractive index of approximately 1.34. When combined with a water-dipping objective lens, the use of such a coverslip can greatly decrease the deterioration of focusing accuracy of a confocal microscope. However, in cytometry, it may be impractical to use a specially-designed objective lens. For example, a cytometer requiring a specially-designed objective lens may prove too costly relative to competing devices or for a given application. Moreover, in some instances, particularly in flow cytometry, it may be impractical to use a coverslip, regardless of the material from which the coverslip is made, because, for example, a flat surface along any side of the flow path may detrimentally affect the orientation or the flow of the analyte through the flow path. Moreover, the use of a specially-designed coverslip, if possible, may prove insufficient to correct aberration in cytometry applications. For example, in some cytometer configurations, such as the flow cytometer described below with respect to FIGS. 3 and 5, elements other than a coverslip, such as the walls of a flow path, may cause focal aberrations.
Regardless of the cause of the focal aberration, the loss of peak intensity and the dispersion of the focus causes a reduction in resolution and in the signal to noise ratio for the collected light or image. As a consequence, light or an image may fail to be resolved, or properties of them may be insufficiently distinct against the background noise of the system. An example of such a property is the fluorescence of a fluorescent-dye labeled cell. A reduction in the amount of light collected from such a cell due to aberration in the AFP 24 may raise the detection threshold for the measurement of such light, and may decrease the precision with which the fluorescent light is measured. This represents a reduction in the efficiency of the optical system as a whole and has practical implications for cytometry and for the design of a cytometry system. By way of example, the implications may require any or all of the following:
Increased observation time for the sample;
Reduced sample rate (analytes per unit time);
Incorporation of more and/or brighter fluorochromes (for fluorescent samples);
More intense excitation light for (to cause fluorochrome excitation);
More sensitive photodetector(s);
Higher numerical aperture of objective lens(es); and
Lower optical and/or electronic background noise.
These requirements may have the effect of increasing the cost of a cytometer, and/or decreasing the throughput or analysis speed of the cytometer, and/or changing the type or expense of fluorochromes, samples, or other components that may be used for a specific purpose in a cytometer.
The situation illustrated in FIG. 2 represents a simple geometry having flat interfaces 20 and 22 between the medium 18 and the cover slip 16. Other geometries may be desirable in the design of a cytometer and may cause additional aberration in the AFP. For example, a flow path having a circular cross section provides desired benefits in a flow cytometer (i.e., a cytometer that measures an analyte as the analyte flows past or through a detection/interrogation region) and, in particular, in a flow cytometer used to sort mammalian sperm cells. Of course, curvature at a boundary between two media having different refractive indices (e.g., an aqueous analyte-bearing medium and a flow path in which the aqueous medium flows) will introduce additional aberration in the light path.
Further, the flow path itself may introduce more than two interfaces between materials with different refractive indices. FIG. 3 illustrates one configuration for a flow cytometer, in which configuration an objective lens 26 is oriented coaxially with a flow 28 bearing an analyte 30. For reasons explained in greater detail in co-pending application Ser. No. 12/495,406, the configuration depicted in FIG. 3 may be preferable over other flow cytometer configurations, especially in situations in which the analyte is a mammalian sperm cell, the viability of which sperm cell must be maintained. As FIG. 3 illustrates, the analyte 30 flows within a flow path 31 toward the objective lens 26, and through a nominal focal point 32, before being diverted by a transverse flow 34 into an exit path 36. Walls 38 and 40 of the flow path 31 may be made of like or dissimilar materials, and typically have a refractive index different from an aqueous solution (not shown) bearing the analyte. The aberration of the AFP in such a situation will be complex due to the fact that rays between the NFP 32 and the objective lens 26 must pass through the walls 38 and 40 of the flow path 31. Current cytometers may avoid this problem, in part, by situating the optical pathway and, in particular, the NFP, such that the walls 38 and 40 of the flow path 31 do not interfere with the optical path.
The use of “water immersion” or “water dipping” objective lenses may, in part, correct aberration caused by the collection of light through a parallel-sided wall or cover slip and/or a fluid. Water immersion objective lenses correct for an optical path that passes through a liquid medium and a determined thickness of a medium of a higher refractive index, typically a glass cover slip. However, even variations in cover slip thickness smaller than the tolerances to which cover slips are typically manufactured can cause the AFP to vary from the NFP. Further, the determined cover slip thickness for which a water immersion objective lens is designed limits the design of any optical cell in which a flow path may be formed. FIG. 4 illustrates a water immersion objective lens 44 having a nominal focal point 32. A tip 45 of the water immersion objective lens 44 sits in water 46 on a glass coverslip 47 of a determined thickness. An aqueous medium 49 (which may be water) having the same refractive index as the water 46 is below the coverslip 47. Contrasted with water immersion objective lenses, water dipping objective lenses are fully corrected for imaging in water without an intervening cover slip.
Moreover, even a cytometer employing a corrected objective lens, such as a water dipping or a water immersion objective lens, remains subject to refractive effects in many instances. For example, FIG. 5 depicts a cytometer 50 in which glass 42 forms the flow path 31. As will be appreciated, the cytometer 50 includes a number of interfaces between different materials, including a glass-immersion fluid interface 51, an immersion fluid-cover material interface 53, a cover material-analyte medium interface 55, and analyte medium-flow path wall interfaces 57 and 59. As in FIG. 4, the tip 45 of the water immersion objective lens 44 sits in water 46. Unlike FIG. 4, the water 46 in FIG. 5 sits on a top thickness 48 of the flow path 31, which top thickness 48 is the same as that of the coverslip 47 depicted in FIG. 4, and corresponds to the thickness for which the water immersion objective lens 44 is corrected. Accordingly, light 52 passing between the water immersion objective lens 44 and the NFP 32 without intersecting the walls 38 and 40 of the flow path 31 remains in focus. However, the glass 42 forming the walls 38 and 40 refracts light 54 that intersects the walls 38 and 40 at the interfaces 57 and 59, causing aberration from the NFP.
It is an objective of the presently described methods and apparatus to mitigate and/or eliminate refractive effects in cytometric devices and methods.